Bill Greenberg

Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation

Abstract Restriction endonucleases Dpn I and Dpn II are produced by two distinct strains of Diplococcus pneumoniae. The two enzymes show complementary specificity with respect to methylation of sites in DNA. From the identity of its cleavage site with that of Mbo I, it appears that Dpn II cleaves at the unmodified sequence 5′-G-A-T-C-3′. Dpn I cleaves at the same sequence when the adenine residue is methylated. Both enzymes produce only double-strand breaks in susceptible DNA. Their susceptibility to Dpn 1 and not Dpn II shows that essentially all the G-A-T-C sequences are methylated in DNA from the pneumococcal strain that produces Dpn II as well as in DNA from Hemophilus influenzae and Escherichia coli. In the dam-3 mutant of E. coli none of these sequences appear to be methylated. Residual adenine methylation in the dam-3 mutant DNA most likely occurs at different sites. Different but characteristic degrees of methylation at G-A-T-C sites are found in the DNA of bacterial viruses grown in E. coli. DNAs from mammalian cells and viruses are not methylated at this sequence. Mitochondrial DNA from Paramecium aurelia is not methylated, but a small proportion of G-A-T-C sequences in the macronuclear DNA of this eukaryote appear to be methylated. Possible roles of sequence-specific methylation in the accommodation of plasmids, in the replication of DNA, in the regulation of gene function and in the restriction of viral infection are discussed.

Horizontal gene transfer to endogenous endophytic bacteria from poplar improves phytoremediation of toluene.

ABSTRACT Poplar, a plant species frequently used for phytoremediation of groundwater contaminated with organic solvents, was inoculated with the endophyte Burkholderia cepacia VM1468. This strain, whose natural host is yellow lupine, contains the pTOM-Bu61 plasmid coding for constitutively expressed toluene degradation. Noninoculated plants or plants inoculated with the soil bacterium B. cepacia Bu61(pTOM-Bu61) were used as controls. Inoculation of poplar had a positive effect on plant growth in the presence of toluene and reduced the amount of toluene released via evapotranspiration. These effects were more dramatic for VM1468, the endophytic strain, than for Bu61. Remarkably, none of the strains became established at detectable levels in the endophytic community, but there was horizontal gene transfer of pTOM-Bu61 to different members of the endogenous endophytic community, both in the presence and in the absence of toluene. This work is the first report of in planta horizontal gene transfer among plant-associated endophytic bacteria and demonstrates that such transfer could be used to change natural endophytic microbial communities in order to improve the remediation of environmental insults.

Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1

The streptococcal plasmid pMV158 and its derivative pLS1 are able to replicate and confer tetracycline resistance in both Gram-positive and Gram-negative bacteria. Copy numbers of pLS1 were 24, 4 and 4 molecules per genome in Streptococcus pneumoniae, Bacillus subtilis and Escherichia coli, respectively. Replication of the streptococcal plasmids in E. coli required functional polA and recA genes. A copy-number mutation corresponding to a 332 base-pair deletion of pLS1 doubled the plasmid copy number in all three species. Determination of the complete DNA sequence of pLS1 revealed transcriptional and translational signals and four open reading frames. A putative inhibitory RNA was encoded in the region deleted by the copy-control mutation. Two putative mRNA transcripts encoded proteins for replication functions and tetracycline resistance, respectively. The repB gene encoded a trans-acting, 23,000 Mr protein necessary for replication, and the tet gene encoded a very hydrophobic, 50,000 Mr protein required for tetracycline resistance. The polypeptides corresponding to these proteins were identified by specific labeling of plasmid-encoded products. The tet gene of pLS1 was highly homologous to tet genes in two other plasmids of Gram-positive origin but different in both sequence and mode of regulation from tet genes of Gram-negative origin.

Genetic and structural characterization of endA: A membrane-bound nuclease required for transformation of Streptococcus pneumoniae*

The endA gene encoding the membrane nuclease of Streptococcus pneumoniae, which is necessary for DNA uptake in genetic transformation, was cloned in a streptococcal vector. This was accomplished by insertional mutagenesis of the gene, cloning of the mutant allele, and substitution of the wild-type allele by chromosomal facilitation of plasmid establishment. Plasmids carrying the endA+ gene complemented cells with endA- in the chromosome to restore DNAase activity and transformability. Determination of its DNA sequence showed the gene to encode a 30 kDa protein, EndA, with a typical signal sequence for membrane transport at its amino end. In vitro synthesis of EndA showed the initial translation product to be enzymatically active without further processing. Comparison with EndA found in cell membranes indicated that the enzyme retained its signal sequence, which apparently anchored the otherwise hydrophilic protein to the membrane. From the nucleotide sequence in the vicinity of endA and the effect of various insertions and deletions, it appears that endA is the last gene in an operon containing at least two other genes. Neither of these upstream genes, nor the downstream gene, are essential for either cell viability or transformability.

Identification of base mismatches recognized by the heteroduplex-DNA-repair system of Streptococcus pneumoniae

The susceptibility to repair of particular base mismatches by the hex system of Streptococcus pneumoniae was examined by comparison of the nucleotide sequence of the wild-type and eight mutant alleles of the malM gene. A detailed restriction map was constructed for pLS70, and the nucleotide sequence was determined for its 3475 bp chromosomal insert, which contains the entire malM gene (encoding amylomaltase), portions of malX and malP (encoding a membrane protein and a phosphorylase, respectively) and a control region. Transition mismatches were highly susceptible to repair; transversion mismatches, much less so. A mismatch caused by a single-nucleotide deletion was reparable, but mismatches with longer deletions were not. The hex system also reduced spontaneous reversion of mutations corresponding to transitions. It is suggested that recognition of donor or nascent DNA strands by the hex system depends on single-strand breaks in the target strand, and that the role of DNA methylation in mismatch repair of Escherichia coli can be accommodated to this model.

Fate of donor DNA in pneumococcal transformation

Abstract The molecular fate of homologous (pneumococcal) and heterologous ( Escherichia coli ) [ 32 P]DNA was examined after introduction into pneumococcal cells. In both cases the composition of 32 P-containing products at the earliest time of observation was the same. About 50% of the incorporated donor material was in the form of single strands, identified by their density in alkaline CsCl gradients. Of the incorporated 32 P, 20 to 30% was in the form of native DNA of pneumococcal composition. Since this was true for donor DNA from E. coli as well as for donor DNA from pneumococcus, it appears that this label entered native DNA by way of de novo synthesis of DNA, presumably from nucleotides. The remaining 32 P in the cell was in the form of dialyzable fragments composed of large amounts of inorganic phosphate and l -α-glycerophosphate and smaller amounts of the four 5′-deoxynucleotides. A plausible origin of labeled inorganic phosphate is the dephosphorylation of nucleotides. The labeled glycerophosphate, in turn, may be derived from inorganic phosphate, since artificially introduced [ 32 P]phosphate rapidly equilibrated with a large pool of intracellular glycerophosphate. Thus, the initial products of entry are envisioned to be equal amounts of single strands and 5′-deoxynucleotides. This would be compatible with their origin in the degradation by a pneumococcal exonuclease of one strand of an incoming DNA duplex. On subsequent incubation, single strands of heterologous origin were slowly degraded; their breakdown products were used for de novo DNA synthesis. The disappearance of homologous single strands was much faster and corresponded to an increase of 32 P in native DNA as well as to the recovery from eclipse of donor marker-transforming activity. It is concluded that single strands are precursors of genetically integrated DNA and that recombination involves the interaction of homologous single strands with host DNA. The ratio of genetically integrated DNA to total DNA incorporated suggests that only half, or somewhat more, of the single-stranded DNA was inserted as intact sequences. Inhibition of DNA synthesis by aminopterin gave results which were compatible with these conclusions.

Single-strand breakage on binding of DNA to cells in the genetic transformation of Diplococcus pneumoniae

Abstract Mutants of Diplococcus pneumoniae that lack a membrane-localized DNAase are defective in transformation because entry of DNA into the cell is blocked. Such mutants still bind DNA on the outside of the cell. The bound DNA is double-stranded and its double-stranded molecular weight is unchanged. Its sedimentation behavior in alkali, however, shows that it has undergone single-strand breakage. The breaks are located randomly in both strands of the bound DNA at a mean separation of 2 × 106 daltons of single-stranded DNA. Both binding and single-strand breakage occur in the presence of EDTA. Single-strand breaks are similarly formed on binding of DNA to normally transformable cells in the presence of EDTA. The single-strand breaks appear to be a consequence of attachment. DNA may be bound to the cell surface at the point of breakage. A mutant that is partially blocked in entry also binds DNA mainly on the outside of the cell. In the presence of EDTA, DNA bound by this mutant undergoes only single-strand breaks. In the absence of EDTA, however, double-strand breaks occur, apparently as a result of the initiation of entry. It is possible that the double-strand breaks arise from additional single-strand breaks opposite those that occurred on binding. The double-strand breaks presumably result from action of the membrane DNAase as it begins to release oligonucleotides from one strand segment while drawing the complementary strand segment into the cell.

ANALYSIS OF A STREPTOCOCCUS PNEUMONIAE GENE ENCODING SIGNAL PEPTIDASE I AND OVERPRODUCTION OF THE ENZYME

The spi gene of Streptococcus pneumoniae was cloned and its nucleotide sequence was determined. It encodes a protein of 204 amino acids that is homologous to bacterial signal peptidase I proteins. The S. pneumoniae protein contains all of the conserved amino acid sequence motifs previously identified in this enzyme from both prokaryotic and eukaryotic sources. Sequence comparisons revealed several additional motifs characteristic of the enzyme. The cloned S. pneumoniae gene complemented an Escherichia coli mutant defective in its leader peptidase gene. Expression of the spi gene in S. pneumoniae appeared to be essential for viability. The cloned gene was shown to produce a polypeptide of approximately 20 kDa. Overproduction of the S. pneumoniae spi gene in an E. coli expression system gave a native protein product, soluble in the presence of a non-ionic detergent, which should be amenable to structural determination.

Regulation of competence for genetic transformation in Streptococcus pneumoniae: expression of dpnA, a late competence gene encoding a DNA methyltransferase of the DpnII restriction system

The chromosomal DpnII gene cassette of Streptococcus pneumoniae encodes two methyltransferases and an endonuclease. One methyltransferase acts on double‐stranded and the other on single‐stranded DNA. Two mRNAs are transcribed from the cassette. One, a SigA promoter transcript, includes all three genes; the other includes a truncated form of the second methyltransferase gene (dpnA) and the endonuclease gene. The truncated dpnA, which is translated from the second start codon in the full gene, was shown to produce active enzyme. A promoter reporter plasmid for S. pneumoniae was devised to characterize the promoter for the second mRNA. This transcript was found to depend on a promoter that responded to the induction of competence for genetic transformation. The promoter contains the combox sequence recognized by a SigH‐containing RNA polymerase. As part of the competence regulon, the dpnA gene makes a product able to methylate incoming plasmid strands to protect them from the endonuclease and allow plasmid establishment. Its function differs from most genes in the regulon, which are involved in DNA uptake. Comparison of R6 and Rx strains of S. pneumoniae showed the temperature dependence of transformation in R6 to result from temperature sensitivity of the uptake apparatus and not the development of competence.

ArsR arsenic-resistance regulatory protein from Cupriavidus metallidurans CH34

The Cupriavidus metallidurans CH34 arsR gene, which is part of the arsRIC2BC1HP operon, and its putative arsenic-resistance regulatory protein were identified and characterized. The arsenic-induced transcriptome of C. metallidurans CH34 showed that the genes most upregulated in the presence of arsenate were all located within the ars operon, with none of the other numerous heavy metal resistance systems present in CH34 being induced. A transcriptional fusion between the luxCDABE operon and the arsR promoter/operator (P/O) region was used to confirm the in vivo induction of the ars operon by arsenite and arsenate. The arsR gene was cloned into expression vectors allowing for the overexpression of the ArsR protein as either his-tagged or untagged protein. The ability of the purified ArsR proteins to bind to the ars P/O region was analyzed in vitro by gel mobility shift assays. ArsR showed an affinity almost exclusively to its own ars P/O region. Dissociation of ArsR and its P/O region was metal dependent, and based on decreasing degrees of dissociation three groups of heavy metals could be distinguished: As(III), Bi(III), Co(II), Cu(II), Ni(II); Cd(II); Pb(II) and Zn(II), while no dissociation was observed in the presence of As(V).